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Proteinase Catalyzed Resynthesis of the Peptide Bond Lys 15ЧAla 16 in the Derivative of Trypsin-Kallikrein Inhibitor (Kunitz) with this Peptide Bond Hydrolyzed.

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I native
CysS?. ... -cqs3*-
J Nal3ll~
- c y s 5 .....
-c y s55 ......
c v s 3 8 - ...................................
reduced and
-cys55.... .
C,J s38--
C y s '...... -(:ys14
I " modified
-c! ys38-
1 . Sephndex G-50
L y s C.OO'.'H,K@
Scheme I
modified ( k * ) inhibitor are different for r-chymotrypsin
(k/k* = 2.2 x lo4)and P-trypsin (kfk*= 50)'*].
Received: J u n e IX. 1974 [ Z 6Xa IE]
revised: July 16, 1974
G e r m a n version: Angcw. Chem. 86,702 11974)
M . J r . a n d R. W Srolocl in P. D. B o w r
Vol. I l l . Academic Press, New York 1971, p. 375.
T h e Enrymes.
[2] H. T\c/rrschc,. Angew. Chem. X6, 21 (1974): Angew. Chem. internat. Edit.
13, 10 (1974)
131 H
K u . w I a n d M. L u d o i d i , Sr., Biochcm. Biophys Res. C o m m u n .
20, 463 ( I 965).
[4] H . T w h ~ ~ , wa/ inrd R. Ohrrmerrr in H. Frrrz a n d I f . Twlre.\chr Proceedings
o f t h e International Research Conferenceon Proteinase Inhibitors. d e Gruyter.
Berlin 1971. p. 135.
[5] L. F Krr.s\ a n d M. La\koizski, Sr., J B i d Chem. 242. 4925 (1967).
L. F K r t , . ~ ,K . A . Wilsori, a n d M. Lu.\koii.skt, Sr., ihid. 246. 3555 (1971).
W K . Liir a n d J. Mrirnhofir, Biochem. Biophys. Rcs. C o m m u n . i i , 467
can be resynthesized with trypsin, chymotrypsin and plasmin.
The long reaction times (half-lives corresponding to 17 weeks"')
due to slow dissociation of the enzyme-inhibitor-complex were
shortened by employing equimolar amounts of trypsin (porcine)
or chymotrypsin (bovine) at pH = 7.8 (0.2M tris(2-hydroxyethy1)ammonium chloride/NaOH, 0.01 M CaCI2) and room
temp. The complexes were dissociated after 1 h (trypsin)
and 48 h (chymotrypsin), respectively, at pH = 1.7 and the
enzyme and inhibitor were then separated on Sephadex G-50
at the same pH. In both cases the sole products'isolated
were the enzyme and the native inhibitor I with intact peptide
bond -F'-F(kinetically controlled dissociation'-"). A
complete resynthesis could also be achieved with catalytic
amounts (20 mol-%%,)
of P-trypsin (bovine) at room temperature
and pH = 3.7 within 24 h (thermodynamically controlled resynthesis).
161 H
TIcltcst~he,H J<vrtry, G. Sdior-p, a n d 7: Drrri in H Ft-rr:. H . Tx+7r,s~hi~,
L J . Griwi~,, a n d E. Trusdwir. Proteinase Inhibitors. 2nd International
Research Conference Bayer Symposium V. Springer, Berlin, in press.
Angew. Chem. 86, 703 (1974). Angew. C'hem.
[7] H . J w i q a n d H. T~ ( ~ lwschr.
internat. Edit. 13, 661 (1974).
[HI L!. Qiru,\r, J . Eriyrl, E . Srel/<w. G M a i r , H. f i c k i ~ s d t r ,a n d H. Jering,
t u r 1. Biochem., in press.
I ::
-C ys-Ly s-Ala-Arg
Proteinase Catalyzed Resynthesis of the Peptide Bond
Lys 15-Ala 16 in the Derivative of Trypsin-Kallikrein
Inhibitor (Kunitz) with this Peptide Bond Hydrolyzed[**]
By Helmut Jcring and Harald Txhrsclw[*]
The open -P,-P;bond (Lys 15, Ala 16)"l in modified
trypsin-kallikrein inhibitor I* (Kunitz) from bovine organs
[*] Priv.-Dor. Dr. H. Tschesche a n d Dr. H. Jering
Organisch-Chemisches Laboratorium,
Lehrstuhl fur Organische Chemie und Biochemie der
Technrschen Universitit
8 Miinchen 2. Arcisstr. 21 (Germany)
[**I F r o m a lecture delivered a t the 2nd International Research Conference
"Proteinase inhibitors"- Bayer Symposium V, Grosse Ledder, October
1973.--Part of the dissertation by H . Jerky.-This work was supported
by t h e Deutsche Forschungsgerneinschaft. W e wish t o thank Fraulein C .
Frnnk for carrying out the a m i n o acid analyses. T h a n k s a r e also d u e to
Bayer AG, Wuppertal for kindly supplying us with T r a s y I o P (trypsin-kallikrein inhibitor (Kunitz) from bovine lung).
Angew. Chem. internat. Edit. I Vol. 13 ( 1 9 7 4 )
No. 10
The structure of the inhibitor with resynthesized peptide bond
was apparent from the following data, which were in accord
with those for the native inhibitor: a) amino acid composition;
b) migration of the homogeneous inhibitor on disc electrophoresis at pH=9.3; c) performic acid oxidation yields only one
polypeptide chain (amino acids 1-58); d ) kinetic constants
of the complex formation with enzyme (inhibition curve)141;
e) difference spectra of the compIexes of enzyme with I and
I*'4J; f) resistance to inactivation with carboxypeptidase B
(2 mol-% in 0.025 M Tris-HCi buffer, pH = 7.2, 24 h at 25 "C).
The resynthesis shows that the equilibrium of the enzymatically
lies comcatalyzed proteolysis of the peptide bond -P,-P;pletely on the side of the native inhibitor. This explains the
failure of attempts to prepare the modified inhibitor by direct
partial proteolysis of the native protein. Furthermore, the
resynthesis of the peptide bond Lys 15-Ala 16 with catalytic
amounts of trypsin, chymotrypsin or plasmin proves that
the reactive site of the inhibitor for these three enzymes is
the same. Thus the trypsin-kallikrein inhibitor (Kunitz) is
to be classified as ~ingle-headed~~,".
of a proteinase (e.g . trypsin) the high association constant
for complex formation(K,,,z 1010-10'4mol/l)can be utilized
for displacing the equilibrium of the carboxypeptidase-catalyzed reaction, i. e. for reincorporation of the residue
We have now used this reaction for insertion of arginine
in position 15 of the modified trypsin-kallikrein inhibitor I*
( K ~ n i t z ) [after
~ ] cleavage of the Lys-15 residue [SOmg I*,
20 units of carboxypeptidase B (Merck) in t m l 0 . 0 2 5 ~
Tris/HCl, pH=7.2, 0.1 M NaCI, 16 h at room temperature].
The modified des-Lys-15 inhibitor can be isolated by ionexchange chromatography. It exhibits a single band in the
disc electrophoresis and due to a lack of one positive charge
it migrates at a slower rate than I*. According to amino
acid analysis it contains exactly one lysine less than I*, and
when subjected to the usual tests['] proves to be inactive
toward trypsin.
Incorporation of arginine was accomplished by dissolving
1.23pmol des-lys-l5 inhibitor, 2.46 p o l trypsin (porcine) and
0.02 pmol carboxypeptidase B (Merck) in 2.4 ml buffer (0.1 M
tris(2-hydroxyethyl)ammonium chloride/NaOH, 0.1 5 M KCl,
0.03 M CaCI2 and 0.01 M NaN3) at pH=6.9 and incubating
the resulting solution with 0.6ml 0.25 M L-arginine in the
same buffer for six days at room temperature. The resulting
trypsin-inhibitor complex was then acidified to pH = 1.7 and
separated into enzyme and inhibitor fractions by molecular
sieve filtration on Sephadex G-50. Ion-exchange on CMSephadex C-25 afforded 0.25 pmol unmodified Arg-15-trypsinkallikrein inhibitor (Kunitz) (Scheme 1).
The Arg-I 5 inhibitor ( l a ) obtained by enzymatic replacement
is characterized by the following data :a) amino acid composition; uniformly migrating band on disc electrophoresis; c)
complete resistance to inactivation by carboxypeptidase B
(2 mol-% for two days), and d) the same inhibitory activity
Received: June 18, 1974 [Z 68b IE]
revised: July 16, 1974
German version. Angew. Chem. 86,703 (1974)
[ I ] H . Jrring and H . Tschesche, Angew. Chem. 86,702 (1974): Angew. Chem.
internat. Edit. 13, 660 (1974).
[2] J . P. Vincenf and M . Lazdunski, Biochemistry 11, 2967 (1972).
[3] W! R. Finkrnstadt and M . Laskowski, Jr., J. Biol. Chem. 242, 771 (1967).
[4] U . Quasf, J . Engel, E. Strfin, G. Mair, H . Tschrschu, and H. Jering,
Eur. J. Biochem., in press.
[ S ] M . Laskowski, J r . and R . W! Srolock in P. D. B o x e r . The Enzymes.
Vol. 111, Academic Press, New York 1971, p. 375.
[6] H. Tschrschu, Angew. Chem. 86,21 (1974): Angew. Chem. internat. Edit.
13, 10 (1974).
Replacement of Lysine by Arginine, Phenylalanine, and
Tryptophan in the Reactive Site of the Trypsin-Kallikrein Inhibitor (Kunitz)t**I
By Helmut Jering and Harald Tschesche[*l
Cleavage of the PI residue from the reactive site of modified
proteinase inhibitors I* with carboxypeptidase B inactivates
the inhibitors"! The cleavage is accompanied by a reduction
in the association constant K,,, (formation of enzyme-inhibitor
complex) of about five to ten orders of magnitude. Reinsertion
D e s - L y s - 1 5- I *
-C ys-X-C 00'
H3N-Ala-A r g
pH =
Arg-15-1 ( l a )
Pl p;
-C ys-X-Ala-A
Phe- 1 5 -1 ( l h )
Trp-15-1 fir)
Scheme 1. For Arg-insertion with trypsin, carboxypeptidase B was used; for Phe- and Trp-insertion
with chymotrypsin, carboxypeptidase A was used. (a), X =Arg; (b), X = Phe; (c). X =Trp.
of the residue P I , e. g . in the reverse reaction of the carboxypeptidase cleavage in the presence of a large excess of free amino
acid PI, increases K,,, by the same amount. In the presence
[*] PIIV.-DOZ.Dr. H. Tschesche and Dr. H. Jering
Organisch-Chemisches Laboratorium,
Lehrstuhl fur Organische Chemie und Biochemie der
Technischen Universitat
8 Miinchen 2, Arcisstr. 21 (Germany)
[**I This work was supported by the Deutsche Forschungsgemeinschaft.
We thank Fraulein C. Frank for carrying out the amino acid analyses.
Thanks are also due t o Bayer AG. Wuppertal for kindly supplying us with
TrasylolO (trypsin-kallikrein inhibitor from bovine lung).
towards the enzymes trypsin, chymotrypsin and plasmin as
the native lysine- inhibitor. Whether differences exist in the
rate constants of association to the enzyme-inhibitor complex
between native and Arg-I 5 inhibitor is at present under investigation. In an analogous reaction sequence with carboxypeptidase A and chymotrypsin under corresponding conditions
we have been able to incorporate the amino acids phenylalanine and tryptophan as residue P,. In contrast to the inhibitors with Lys or Arg in the reactive site, the homologous
Phe and Trp-inhibitors ( Z b ) and ( I c ) are poor trypsin but
good chymotrypsin inhibitors. Corresponding results were
obtained with the soybean inhibitor (Kunitz)['. 31.
Angew. Chem. internat Edit. J Vol. 13 ( 1 9 7 4 ) J No. 10
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hydrolyze, bond, 15чala, resynthesis, trypsin, kallikrein, kunitz, derivatives, catalyzed, lys, inhibitors, proteinase, peptide
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